The overall goal of this project is (1) to evolve in situ RT-PCR to the point where it can become routine for basic, epidemiological and cancer research and (2) increase the throughput of in situ RT-PCR to conduct whole genome profiling of human tumor viruses (and eventually multi-gene arrays of human oncogenes). To achieve uniformity of performance, we rely on our previously validated real-time primer set for Kaposi's sarcoma-associated herpesvirus (HHV-8/KSHV) (Dittmer, Cancer Research 63:2010-15 (2003)), which exhibits extremely uniform amplification efficiencies for each primer pair. We will test the appropriateness of this 82 primer set for in situ real-time RT-PCR of tumor sections; we will compare--on the same sample--whole tumor transcription profiling to in situ analysis for all mRNAs in the viral genome; and we will validate both procedures relative to viral protein expression. Even though this R03 application uses KS and KSHV primarily as a test case to validate this technology, looking at KS and KSHV transcription this study in itself will yield valuable information about the biology of this disease, because tumor heterogeneity is one of the defining characteristics of KS. In KS lesions tumor progression depends on and responds to viral gene expression. This study will provide a road map for a better understanding of KS biology and clinical classification of KS tumors.